Oligonucleotides are polymers of nucleotides and are formed by sequentially linking a number of nucleotides together in a chain. These chains may have less than twenty nucleotides, but in some cases may have several hundred nucleotides. Oligonucleotides are useful for, inter alia, hybridizing to complementary DNA or RNA. The interactions between oligonucleotides and modified oligonucleotides with complementary RNA is being exploited in a number of therapeutic, diagnostic, and research applications. Consequently, oligonucleotides may be useful in, for example, pharmaceutical research, as well as for certain medical testing.
Oligonucleotides may be synthesized in a laboratory environment using a series of steps. During this synthesis, nucleotides are attached in a chain in a desired order. The series of steps taken when adding a nucleotide is referred to as a synthetic cycle, and may involve: removal of a dimethoxytrityl group (detritylation); attachment of the desired nucleotide phosphoramidite to the location previously held by the dimethoxytrityl group (coupling); blocking of any sites where the dimethoxytrityl group was not replaced by the desired nucleotide phosphoramidite (capping); and stabilizing the resultant structures (oxidation). These steps may be conducted manually, or by automatic synthesizers that have been developed that facilitate the process.
Essentially, each of the steps in the synthetic cycle involves a reagent that reacts with the oligonucleotide undergoing synthesis. For the synthetic cycle described above, a different reagent may be used in each of the steps of detritylation, coupling, capping and oxidation. It will be appreciated that for each nucleotide that is added to the chain, numerous reagents may be used. To provide more efficient reactions during the synthesis, it is desirable to reduce the degree to which reagents are allowed to mix with other reagents used in adjacent steps.
The synthesis may be conducted in a laboratory using a type of reactor called a synthesizer. Synthesizers include a reaction zone in which the synthesis occurs, and this reaction zone typically includes a frit, or filter, at the top and bottom of the reaction zone. A reagent flows through the top frit and into the reaction zone where the reaction takes place. The excess reagent and any products of the reaction then exit the reaction zone through the bottom frit. By introducing reagents serially through the top frit and removing them through the bottom frit, the degree of interaction between the reagents may be decreased as compared to earlier systems.
Oligonucleotide synthesis typically involves the use of a solid support, which may consist of a granular polymeric material, to which the first nucleotide is attached; other nucleotides then are added sequentially to form a chain. Following the addition of the final nucleotide, the chain is separated from the solid support. The solid support provides a foundation on which the oligonucleotide may be built, and the larger size of the solid support helps to avoid the loss of the oligonucleotide chain as different reagents are processed through the synthesizer during the synthetic cycles. In this regard, the granules of the solid support typically are larger than the pores of the bottom frit, so that the solid support does not exit the reaction zone as the excess reagents flow through the reaction zone and out the bottom frit.
In a reaction zone containing a solid support, there may exist a space between the solid support and the top frit. This space, called the “headspace,” provides a volume in which a reagent is able to mix with the reagent used in the preceding step in the synthesis process. In order to reduce this interaction, additional solid support may be used to fill the volume of the reaction zone and thereby reduce the volume available for mixing of the reagents.
Nevertheless, some solid supports may swell or contract when exposed to different reagents during the synthetic process. Thus, the volume of the solid support undergoing the reactions varies during the synthetic process. Some previously-known synthesizers have attempted to address this variation by varying the axial compression applied by the top frit. In such systems, the top frit is attached to a piston which moves up and down within the reaction zone of the synthesizer. In such a system, a predetermined pressure is maintained hydraulically by adjustment of the location of a hydraulic piston within the reaction zone.
The foregoing method of adjusting the top frit location does not provide for optimal conditions within the reactor for synthesizing oligonucleotides involving dozens of reactions and significant changes in pressure, volume and other conditions. Additionally, because numerous, vastly different pressures may occur during oligonucleotide synthesis, there may be no direct relationship between the pressure within the reaction zone and the amount of headspace that forms during any given step. Accordingly, previously-known systems are unable to provide oligonucleotide synthesis while also minimizing or eliminating headspace.
Moreover, while headspace may be reduced by applying a sufficiently large pressure to a system designed to maintain a constant pressure, such a pressure would need to be at least as large as the maximum pressure observed during the synthesis process. In a synthesis process involving a wide range of pressures, however, the continued application of high pressure during a step involving only a low pressure may cause undesirable compression of the solid support. Accordingly, the effectiveness of the system may be compromised and the desired reactions may not occur as desired.
Accordingly, there is a need for apparatus and methods for synthesizing oligonucleotides that facilitate efficient reactions by reducing or eliminating the headspace.
There is also a need for apparatus and methods for synthesizing oligonucleotides that reduce or eliminate headspace without causing undue compaction of the contents of the synthesizer.
There is yet further a need for apparatus and methods for synthesizing oligonucleotides that reduce or eliminate headspace for a system that experiences dynamic changes in pressure and/or volume when undergoing multiple reactions.